The Saccharomyces cerevisiae mating pheromone a-factor is an extracellular signaling molecule whose biogenesis pathway is distinctive. The a-factor precursor undergoes post-translational addition of an isoprene lipid (farnesyl), methylation, and several proteolytic processing steps to produce a prenyl- and methyl-modified oligopeptide. a-Factor does not exit cells via the classical secretory pathway which involves intracellular secretory organelles, but instead utilizes a novel membrane transport pump for export. Several components of the a-factor biogenesis machinery are known: the RAM1 and RAM2 gene products together comprise the farnesyltransferase which mediates a-factor prenylation, STE14 encodes the methyltransferase, and STE6 encodes the a-factor export pump. The overall goal of this proposal is to gain a high resolution view of a-factor biogenesis by investigating modification, processing, and secretion of a-factor in detail and examining how these events are co-ordinated. Molecular, genetic, and biochemical approaches will be used to probe the activity of known biogenesis components, genetically identify new components, and examine protein-protein interactions between a-factor and its biogenesis machinery. Specific aims are: 1) to identify recognition determinants in a-factor by mutational analysis; 2) to examine the extent to which farnesyl is a specific biogenesis determinant by testing whether related lipids can substitute for it in a-factor; 3) to ascertain the cellular location of the STE14 methyltransferase, and further examine its role in a-factor biogenesis; 4) to genetically identify the genes involved in N-terminal processing of a-factor; 5) to determine residues within the STE6 transporter that are critical for a-factor recognition; 6) to characterize a novel a-factor-related peptide (AFRP) that exits cells by a STE6-independent pathway; and 7) to use heterologous systems to examine the biogenesis of a-factor and related pheromones produced by other yeast species. The study of a-factor will provide insight into a variety of cellular mechanisms that are medically relevant including prenylation and methylation of Ras oncoproteins, efflux of chemotherapeutic drugs by the mammalian multidrug resistance protein ( a close relative of the a-factor transporter STE6), and processing and membrane traversal of peptides involves in antigen presentation by the immune system.